Abstract

G protein-coupled receptor kinase interactors (GITs) regulate focal adhesion (FA) turnover, cell spreading, and motility through direct interaction with paxillin and the Rac-exchange factor Pak-interacting exchange factor beta (betaPIX). However, it is not clear whether GITs function to activate or repress motility or if the predominant GIT forms, GIT1 and GIT2, serve distinct or redundant roles. Here we demonstrate an obligatory role for endogenous GIT2 in repression of lamellipodial extension and FA turnover by Rac1- and Cdc42-dependent signaling pathways, respectively. Moreover, we show that the SH2-SH3 adaptor protein Crk is an essential target of GIT2 inhibition. Unexpectedly, we find that betaPIX is dispensable for the effects elicited by knockdown of GIT2. Finally, we show that loss of GIT2 is sufficient to induce migration of the nontransformed epithelial cell line MCF10A. These results suggest that inactivation of GIT2 function is a required step for induction of cell motility and that GIT2 may be a target of oncogenic signaling pathways that regulate cell migration.

GIT1–βPIX–PAK complexes are preserved and GIT1 remains associated with FCs in GIT2 knockdown cells. (A–F) Control, GIT1-kd, and GIT2-kd cells were stained for GIT1 or GIT2. (G) HeLa cells were formaldehyde crosslinked and lysates were prepared as described in Materials and methods. Lysates were immunoprecipitated with GIT1- and GIT2-specific polyclonal antibodies. Following reversal of crosslinks, immunoprecipitates were blotted for paxillin, β-catenin, vimentin, and GIT1&2. (H) Crosslinked lysates from control or GIT2-kd cells were immunoprecipitated with GIT1 and GIT2 antibodies and blotted for paxillin, vimentin, and GIT1. (I, J) Control, GIT1-kd and GIT2-kd cells were lysed without formaldehyde crosslinking. (I) Lysates were immunoprecipitated with GIT1- and GIT2-specific polyclonal antibodies and blotted for βPIX, GIT1, and GIT2. (J) Lysates from knockdown cells were immunoprecipitated with anti-PAK antibodies and blotted for GIT1, GIT1&2, or βPIX. Scale bar: 20 μM.

GIT2 represses Rac1 and Cdc42-dependent membrane ruffling and FA turnover, respectively. (A–D) HeLa cells were transfected with GIT2 shRNA with or without myc-tagged Cdc42(N17). Cells were stained for paxillin (red) and myc (green). (E–L) HeLa cells were transfected with GIT2 shRNA with or without myc-tagged Rac1(N17). Cells were stained for paxillin (red) and myc (green) (E–H), or for F-actin (red) and myc (green) (I–L). (M–Q) HeLa cells were transfected with control or Rac1 siRNA together with control or GIT2 shRNA. Cells were lysed and blotted for Rac1, GIT2, and ERK (M), or stained with phalloidin (green) and paxillin (red) (N–Q). Scale bar: 20 μM.

GIT2 represses migration of MCF10A cells. MCF10A cells stably expressing GIT2 shRNA were blotted for GIT2 (A) and stained for β-catenin in green and F-actin in red (B, C). (D, E) To quantify changes in motility, control and GIT2 knockdown cells were harvested and added to the top chamber of either uncoated or MATRIGELTM-coated transwell filters. The number of cells migrated to the bottom chamber is expressed as a percentage of the total cells plated. Color panels in panel E show staining of migrated cells with crystal violet. Values are means±s.d. (n=5 filters) from a representative experiment. Scale bar: 20 μM.